Integrated Metabolomic and Transcriptomic Analysis of the Anthocyanin and Proanthocyanin Regulatory Networks in Red Walnut Natural Hybrid Progeny Leaves

BackgroundWalnuts are one of the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut type, red walnut ‘RW-1’, with red leaves and seed coats was selected as the plant material because of its higher anthocyanin and proanthocyanin (PA) contents. Anthocyanins and PAs coprise important secondary defense methods for plants to respond to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts.ResultsFrom the results of widely targeted metabolome and anthocyanidin detection analysis, 395 substances, including 4 PAs and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all anthocyanin types in SR were significantly upregulated compared with SG. Additionally, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified as the primary components of anthocyanidins because of their higher contents. Nine anthocyanidins, malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside), were detected only in the SR walnuts. For PAs, proanthocyanin C1 was upregulated in SR compared with SG, while proanthocyanin B1 and proanthocyanin B3 were upregulated in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H, UFGTs, LAR and ANR), four MYBs and six WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnut.ConclusionsOur results provide valuable information on anthocyanin and PA metabolites and candidate genes in anthocyanin and PA biosynthesis, which provides new insights into anthocyanin and PA biosynthesis in walnuts.

Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in red walnut

Background Walnut is one of the most important dry fruit crops worldwide, typically green leaves and yellow-brown or gray-yellow seed coats. A specific walnut type, red walnut ‘RW-1’ with red leaves and seed coats was selected as plant material because of higher anthocyanins contents. Anthocyanins are important colorants with strong antioxidant activity, especially, benefic for human health. However, few studies focused on the molecular mechanism of anthocyanin biosynthesis in walnut. Results From the results of Widely Targeted Metabolome and anthocyanidin detection analysis, 395 substances, including 4 procyanidins and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all the anthocyanins in SR were significantly up-accumulated comparing with SG. Also, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified to the primary components of anthocyanidins because of the higher contents. It was noted that 9 anthocyanins including malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside) were detected only in SR walnut. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H and UFGTs), and four MYBs in anthocyanin biosynthetic pathway were significantly higher in SR walnut. Conclusions We identified the color formation of SR leaves is due to the accumulation of anthocyanins. And our results obtained the valuable information on the anthocyanin metabolites and candidate genes in anthocyanin biosynthesis, which provided new insights into the anthocyanin biosynthesis in walnuts.

Fine-Mapping And Identification of A Candidate Gene Controlling Seed Coat Color In Melon (Cucumis Melo L. Var. Chinensis Pangalo)

Seed coat color is related to flavonoid content which is closely related to seed dormancy. According to the genetic analysis of a six-generation population derived from two parents (IC2508 with a yellow seed coat and IC2518 with a brown seed coat), we discovered that the yellow seed coat trait in melon was controlled by a single dominant gene, named CmBS-1. Bulked segregant analysis sequencing (BSA-Seq) revealed that the gene was located at 11,860,000–15,890,000 bp (4.03 Mb) on Chr 6. The F2 population was genotyped using insertion-deletions (InDels), from which cleaved amplified polymorphic sequence (dCAPS) markers were derived to construct a genetic map. The gene was then fine-mapped to a 233.98 kb region containing 12 genes. Based on gene sequence analysis with two parents, we found that the MELO3C019554 gene encoding a homeobox protein (PHD transcription factor) had a nonsynonymous single nucleotide polymorphism (SNP) mutation in the coding sequence (CDS), and the SNP mutation resulted in the conversion of an amino acid (A→T) at residue 534. In addition, MELO3C019554 exhibited lower relative expression levels in the yellow seed coat than in the brown seed coat. Furthermore, we found that MELO3C019554 was related to 12 flavonoid metabolites. Thus, we predicted that MELO3C019554 is a candidate gene controlling seed coat color in melon. The study lays a foundation for further cloning projects and functional analysis of this gene, as well as marker-assisted selection breeding.

Comparative transcriptome and flavonoids components analysis reveal the structural genes responsible for the yellow seed coat color of Brassica rapa L.

Background Seed coat color is an important horticultural trait in Brassica crops, which is divided into two categories: brown/black and yellow. Seeds with yellow seed coat color have higher oil quality, higher protein content and lower fiber content. Yellow seed coat color is therefore considered a desirable trait in hybrid breeding of Brassica rapa, Brassica juncea and Brassica napus. Methods Comprehensive analysis of the abundance transcripts for seed coat color at three development stages by RNA-sequencing (RNA-seq) and corresponding flavonoids compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out in B. rapa. Results We identified 41,286 unigenes with 4,989 differentially expressed genes between brown seeds (B147) and yellow seeds (B80) at the same development stage. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 19 unigenes associated with the phenylpropanoid, flavonoid, flavone and flavonol biosynthetic pathways as involved in seed coat color formation. Interestingly, expression levels of early biosynthetic genes (BrCHS, BrCHI, BrF3H, BrF3’H and BrFLS) in the flavonoid biosynthetic pathway were down-regulated while late biosynthetic genes (BrDFR, BrLDOX and BrBAN) were hardly or not expressed in seeds of B80. At the same time, BrTT8 and BrMYB5 were down-regulated in B80. Results of LC-MS also showed that epicatechin was not detected in seeds of B80. We validated the accuracy of our RNA-seq data by RT-qPCR of nine critical genes. Epicatechin was not detected in seeds of B80 by LC-MS/MS. Conclusions The expression levels of flavonoid biosynthetic pathway genes and the relative content of flavonoid biosynthetic pathway metabolites clearly explained yellow seed color formation in B. rapa. This study provides a foundation for further research on the molecular mechanism of seed coat color formation.

Cinnamoyl-CoA Reductase 1 (CCR1) and CCR2 Function Divergently in Tissue Lignification, Flux Control and Cross-talk with Glucosinolate Pathway in Brassica napus

Cinnamoyl-CoA reductase (CCR) is the entry point of lignin pathway and a crucial locus in dissection and manipulation of associated traits. Brassica crops have worldwide importance, but their CCR-related metabolisms and traits are largely uncharacterized. Here, 16 CCR genes are identified from B. napus and its parental species B. rapa and B. oleracea. They are divided into CCR1 subfamily and CCR2 subfamily, which differ from each other in organ-specificity, participation in yellow-seed trait and responses to various stresses. BnCCR1 is preferentially involved in biosynthesis of G and H lignins and development of vascular system, while BnCCR2 is preferentially involved in biosynthesis of S lignin and development of interfascicular fibers. BnCCR1 has stronger effects on lignification-related development, lodging resistance, flux control and seed color, whereas BnCCR2 has stronger effect on sinapates biosynthesis. BnCCR1 overexpressing plants show a delay in bolting and flowering, while BnCCR2 overexpressing plants have less developed vascular system in leaf due to suppressed G lignin accumulation. Unexpectedly, both BnCCR1 and BnCCR2 overexpressors show no improvement in resistance to UV-B and S. sclerotiorum. Besides, their glucosinolate profiles are greatly and almost oppositely remodeled through pathway crosstalk. These results provide systemic dissection on Brassica CCRs and CCR1-CCR2 divergence in Brassicaceae.

Screening and genetic studies on resistance to Soil-born Cereal Mosaic Virus (SBWMV) in rye

Due to several reasons soil-borne viruses such as the furoviruses, i. e., cereal mosaic virus (SBCMV) and wheat mosaic virus (SBWMV) as well as the bymovirus wheat spindle streak mosaic virus (WSSMV) gained importance in cereal breeding including rye. High yield losses are recorded, today. Since there is no or little resistance to these viruses in modern rye cultivars, an extended screening for resistance was initiated. In addition to earlier screenings, 37 rye genotypes were tested for resistance. Among them, three genotypes were found with persistent resistance to SBCMV. They belong to Secale montanum and S. vavilovii species, i. e., wild types of rye. One accession, PC2243 (S. montanum), was used as a resistance donor for the present genetic study. In F2 generation, it was observed that resistance to SBCMV is independently inherited from WSSMV. The evaluation of the ELISA values pointed to a 3:1 distribution assuming duplicate dominant epistasis. Molecular marker analysis supports this segregation pattern. By composite interval mapping a QTL on chromosome 2R could be detected. It can be assumed that there is a DNA region of about 13 cM on the long arm of chromosome 2R (2RL) harboring SBCMV resistance with the closest markers “C9654_1947” and “isotig11640”. Moreover, genotypes with a yellow seed coat showed practically no infection with SBCMV. Thus, the resistance gene could be linked to the allele an1 determining non expression of anthocyanins. This locus was also mapped earlier on chromosome 2R.

Development of a Yellow-Seeded Stable Allohexaploid Brassica Through Inter-Generic Somatic Hybridization With a High Degree of Fertility and Resistance to Sclerotinia sclerotiorum

The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum, Alternaria brassicae, pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot.

CDC Dorado yellow seed coat oilseed flax

CDC Dorado is a yellow seed coat oilseed flax (Linum usitatissimum L.), registered in 2017 by the Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan. This cultivar has 7% higher yield than AC Nugget in the Black soil zone of the Prairies coupled with a maturity earlier than Flanders across ten site years in Western Canada. It has a medium (45.2%) oil content, high iodine value (IV 204.2) and alpha-linolenic acid (ALA) content (64%), and a thousand seed weight (TSW) of 6.0 g. It has disease ratings of immune to flax rust caused by Melampsora lini and is moderately resistant to wilt and powdery mildew caused by Fusarium oxysporum f. sp. lini and Oidium lini, respectively.

CDC Melyn yellow seed coat oilseed flax

CDC Melyn is a yellow seed coat oilseed flax (Linum usitatissimum L.), registered in 2016 by the Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan. This cultivar has yield (106%) comparable to cultivar AC Nugget (the yield standard for yellow seed coat oilseed flax) and maturity rating equal to Flanders. It has a medium (46.6%) oil content, iodine value (199.4), alpha-linolenic acid content (61%) and a thousand seed weight of 5.0 g. It is immune to flax rust caused by Melampsora lini and moderately resistant to wilt and powdery mildew caused by Fusarium oxysporum f. sp. lini and Oidium lini, respectively.

Chemical Profiling of Roasted Mustard Oils of Khokana, Nepal

Background: Khokana, commonly known as “the living museum” of Nepal is famous for “the roasted mustard oil”. People have been using oil for a long time ago and it is trusted that roasted mustard oil has many health benefits. Detail chemical profiling of roasted mustard oil of Khokana has not been reported yet. Objectives: Detail chemical profiling of roasted mustard oil and chemical variations in different seeds available for roasting. Methods: Three different roasted mustard oils (Nepali, Indian, and other origins seeds) were taken for chemical profiling of oil. The GC/MS of all samples was analyzed by the gas chromatography-mass spectrometer Shimadzu GCMS-QP2010 Ultra. Results: The GC/MS of all samples were carried out and the GC-MS analysis revealed that Nepali (brown seed) and other origins (yellow seed) sample showed erucic acid as a major compound with almost 40-50%. Nepali oil showed gamma-tocopherol (<1%) which is a potent antioxidant. Whereas Indian mustard (black seed) oil showed cis-oleic acid as a major compound with 50-60% and Erucic acid was below 1% in Indian seed oil.